Method of producing a binder-toxin fusion protein in a plant cell or a whole plant

Abstract

The present invention relates to a method of producing a binder-toxin fusion protein comprising at least, one protein binder selected from the group consisting of an antibody, an antibody fragment or derivative retaining target binding capacity, or an antibody mimetic, optionally, a peptide linker, and at least one protein toxin or protein protoxin. The method comprises the steps of: contacting a plant cell or a whole plant with a nucleic acid construct comprising in operational linkage at least the following (A) at least one polynucleotide encoding for the protein binder, or a target binding chain or domain thereof, and either B1) a polynucleotide encoding for a cleavable peptide linker and a polynucleotide encoding for a protein toxin, or B2) a polynucleotide encoding for a protein protoxin, which protoxin comprises a cleavable domain for activation thereof, allowing the construct to integrate into the nucleus of the plant cell, or of one or more cells of the whole plant, and expressing the fusion protein encoded by the nucleic acid construct.

1 . A method of producing a binder-toxin fusion protein comprising at least:
 a) one protein binder selected from the group consisting of
 an antibody 
 an antibody fragment or derivative retaining target binding capacity, and 
 an antibody mimetic, 
   b1) a cleavable peptide linker and a protein toxin, or   b2) a protein protoxin comprising a cleavable domain,   
       said method comprising:
 (i) contacting a plant cell or a whole plant with a nucleic acid construct comprising in operational linkage at least the following
 A) at least one polynucleotide encoding for the protein binder, or a target binding chain or domain thereof, and either
 B1) a polynucleotide encoding for a cleavable peptide linker and a polynucleotide encoding for a protein toxin, or 
 B2) a polynucleotide encoding for a protein protoxin comprising a cleavable domain for activation thereof, 
 
 
 (ii) allowing the construct to integrate into the nucleus of the plant cell, or of one or more cells of the whole plant, and 
 (iii) expressing the fusion protein encoded by the nucleic acid construct, 
 
       wherein the peptide linker or the cleavable domain in the protoxin is not cleavable by an enzyme expressed by the plant cell, or an enzyme that is produced by the plant host. 
     
     
         2 . The method according to  claim 1 , which further comprises
 (iv) recovering and/or purifying the fusion protein expressed in (iii).   
     
     
         3 . The method according to  claim 1 , wherein the plant or plant cell is from the genus  Nicotiana.    
     
     
         4 . The method according to  claim 1 , wherein the peptide linker or the cleavable domain in the protoxin is specifically or non-specifically cleavable by an enzyme expressed by a mammalian cell, or an enzyme that is produced by a mammalian host. 
     
     
         5 . The method according to  claim 1 , wherein the cleavable linker or the cleavable domain in the protoxin comprises at least one cleavage site selected from the group consisting of
 a) Endosomal and/or Lysosomal proteases cleavage site   b) Cytosolic protease cleavage site, and   c) Cell surface proteases cleavage site.   
     
     
         6 . The method according to  claim 1 , wherein at least one protein toxin or protoxin is an enzyme. 
     
     
         7 . The method according to  claim 1 , wherein, the protein toxin is at least one selected from
 (1) Cell death inducing proteins,   (2) Protein synthesis inhibitors,   (3) Membrane perturbating proteins, and   (4) Cell division inhibiting proteins.   
     
     
         8 . The method according to  claim 1 , wherein the protein toxin is a mammalian toxin, optionally selected from the group of Granzymes, optionally Granzyme B, or a fragment thereof that retains toxic activity of said protein toxin. 
     
     
         9 - 19 . (canceled)